In case of the production of recombinant proteins in heterologous expression systems like e.g. Escherichia coli, these proteins often form inactive, insoluble aggregates (so-called “retractile bodies” or “inclusion bodies”). Additionally, these inclusion bodies are contaminated by host cell components like host cell proteins, nucleic acids, endotoxins and low molecular weight contaminants. It is assumed that the formation of these inclusion bodies is a result of the very high local concentration of the heterologous protein in the cell during induction and protein biosynthesis. However, the primary amino acid sequence of the heterologous protein in question is also of great importance as well as the presence of cysteine-residues that form covalent disulfide bonds during oxidative refolding. Before these target proteins can be used, e.g. for therapeutic purposes, the inclusion bodies have to be purified and, subsequently, the three-dimensional structure has to be renatured to convert the protein into the biologically active conformation.
A commonly applied sequence of process steps involves, first, the solubilization of the inclusion bodies by the addition of high concentrations of chaotropic, denaturing agents (e.g. guanidinium hydrochloride or urea), or by the addition of strongly acidic agents like, e.g. glycine/phosphoric acid mixtures. Concurrently, intramolecular disulfide bonds present in the inclusion bodies may be either reduced chemically or cleaved by the so-called sulfitolysis procedure involving sulfite and an oxidizing agent. Secondly, the solubilized protein mixture may be further purified by either chromatographic means or filtration methods, both of which are well known procedures for those skilled in the art.
Subsequently, the linearized, monomeric protein solution in the presence of high concentrations of chaotropic agent is highly diluted in order to allow for the formation of the biologically active form. This can be performed either rapidly (by simple dilution into a large volume of refolding buffer) or slowly by diafiltration or by dialysis against the refolding buffer. Other techniques described in the literature involve the adsorption of the target protein onto a chromatographic resin and, subsequently, lowering the concentration of chaotropic agent allowing refolding to take place, or size exclusion chromatography in order to separate the protein chains thereby circumventing the tendency to form aggregates. In every case, the concentration of the chaotropic salt has to be decreased below a certained limit, which is dependent on the target protein, e.g. usually below 0.5 M guanidinium hydrochloride.
The major side reaction during refolding is the formation of insoluble aggregates, which is dependent on the local concentration of folding intermediates. In the literature, a broad range of folding aids are described, effectively suppressing this formation of insoluble protein aggregates, like e.g. chaperone proteins, other types of proteins (e.g. bovine serum albumin), and several types of non-protein materials, including sugars and cyclic sugars, short chain alcohols like e.g. glycerol, pentanol, hexanol, enzyme substrates, synthetic polymers, detergents, and chaotropic salts (de Bernardez Clark, E (1998): Curr. Opinion Biotechnol. 9: 157-163 and citations therein; Lilie H, Schwarz E, Rudolph R (1998): Curr. Opinion Biotechnol. 9: 947-501 and citations therein; Sharma A, Karuppiah N (1998): U.S. Pat. No. 5,728,804 filed Jun. 2, 1995). A different approach has recently been published where so-called artificial chaperones are used to keep hydrophobic folding intermediates in solution (Gellmnan S, Rozema D B (1996): U.S. Pat. No. 5,563,057 filed Oct. 31, 1994). In a first step, hydrophobic folding intermediates are trapped into detergent micelles leading to a suppression of protein aggregation. The trapped folding intermediates cannot fold to the native conformation. In a second step, a “stripping agent”, like e.g. different cyclodextrins or linear dextrins, are added in considerable molar excess to the remove the detergent again allowing the protein to refold into its biologically active conformation. There are several drawbacks to this approach like 1. Large molar excess of the expensive “stripping agent”, 2. Protein aggregation occurring during the “stripping” phase, 3. Difficulty to remove residual detergent bound to the target protein, 4. Limitations in protein capacity and solubility of cyclodextrins and 5. Sensitivity of the artificial chaperone system with respect to process variations (limited robustness). Moreover, artificial chaperone systems are specific with respect to the target protein, the type of detergent and “stripping agent” and the experimental conditions employed. Hence, there is no generic artificial chaperone system available (Daugherty D L, Rozema D, Hanson P E, Gellman S H (1998): J. Biol. Chem. 273: 33961-33971; Rozema D, Gellman SH (1996): J. Biol. Chem. 271: 3478-3487).
Most of the above mentioned aggregation suppressors only work with a limited number of proteins. One exception is the amino acid L-arginine, which was shown to be generally applicable to a wide range of different proteins like e.g. t-PA, Fab fragments, lysozyme and other enzymes (Rudolph R, Fischer S, Mattes R (1997): Process for the activating of gene-technologically produced, heterologous, disulfide bridge-containing eukaryotic proteins after expression in prokaryotes. U.S. Pat. No. 5,593,865; Rudolph R, Pischer S, Mattes R (1995): Process for the activation of T-PA or ING after genetic expression in prokaryotes. U.S. Pat. No. 5,453,363; de Bernardez Clark, E (1998): Curr. Opinion Biotechnol. 9: 157-163 and citations therein).
L-arginine was shown for a number of proteins to be effective only in high molar excess with respect to the molarity of the protein to be refolded. The mechanism by which L-arginine suppresses the formation of protein aggregates is still unknown (Lilie H et al. (1998): Curr. Opinion Biotechnol. 9: 497-501). Moreover, L-arginine is an expensive, chiral fine chemical.
Hence, there is still a need to develop strategies for protein refolding using conventional techniques. From the state of the art, no generally useable, chemically simple and inexpensive aggregation suppressor is known, which can be applied in a commercially attractive refolding process of proteins at high concentrations of up to 0.5-1 g/L.